Our general objective is to characterize the functional organization of the bed nucleus of the stria terminalis (BNST), a brain region involved in anxiety but about which little is known. In the process, we aim to shed light on the mechanisms underlying fear generalization, a hallmark of anxiety disorders. It is commonly believed that BNST generates long lasting anxiety-like states in response to diffuse contingencies but that it is not involved in the expression of learned fear responses to discrete sensory cues, the latter depending on the amygdala. In contrast, our previous work indicates that BNST activity contributes to cued fear in two ways: by prolonging fear responses long after the threatening stimulus has ended (temporal generalization of fear) and by allowing different (safe) cues to also trigger fear (stimulus generalization of fear). Since experiencing fright long after the threa has passed or in response to safe stimuli are hallmarks of anxiety disorders, understanding how BNST contributes to fear generalization is an issue of considerable translational significance. Thus, this proposal will examine how BNST, via its reciprocal connections with the amygdala and projections to brainstem fear effectors, contributes to the generalization of learned fear responses. However, before addressing this question, we need to improve our understanding of the basic physiological organization of BNST. Indeed, BNST is known to contain multiple physiological cell types, expressing different neurotransmitters, and projecting to various sites that influence fear expression. However, how these various properties correlate with each other is unknown. Thus in Aims #1-2, we will first strive to obtain a morpho-physiological wiring diagram of BNST by combining patch recordings of retrogradely labeled BNST cells in vitro, biocytin labeling, photic uncaging of glutamate, and post-hoc immunofluoerescence for GABAergic and glutamatergic markers. As a result, will be able to assign cells recorded in vivo (Aim #3) to locations in this circuit based on their physiology. In Aim #3, guided by the data obtained in Aims #1-2, we will perform extracellular recordings of rat BNST and amygdala neurons. The rats will be subjected to a differential fear conditioning paradigm that reproduces the inter-individual variations in fear responding seen in humans. The projection site of recorded cells will be identified by antidromic invasion. By relating the unit data with inter-individual variations in fear responding, we will formulate testable predictions regarding the mechanisms underlying the temporal and stimulus generalization of fear. Last, in Aim #4, we will test these predictions by selectively inhibiting or activating particular BNST or amygdala outputs using in vivo optogenetic inhibition or stimulation. Given that similar networks underlie fear learning in animals and humans, the proposed studies might shed light on the pathophysiology of anxiety disorders. PUBLIC HEALTH RELEVANCE: The ability to associate fear responses to new stimuli or circumstances on the basis of experience is necessary for survival. On the other hand, excessive fear can lead to a pernicious cycle of avoidance that gradually spreads and intensifies, as seen in anxiety disorders. Much data suggest that research on fear learning mechanisms constitutes our best hope of understanding anxiety disorders. Indeed, similar networks underlie fear learning in animals and humans (Phelps & LeDoux '05). Moreover, these networks show abnormal activity patterns in humans with anxiety disorders (Bremner '08). Thus, to shed light on the pathophysiology of these disorders, we must further our understanding of fear learning networks. This proposal aims to do just that, focusing on the bed nucleus of the stria terminalis (BNST), a relatively neglected, yet critical, node in the fear circuit. !